1. Field of the Invention
The present invention relates to the use of Time-of-Flight secondary ion mass spectrometry (TOF-SIMS) for the creation of a diagnostic screening method for Smith-Lemli-Opitz (SLO) Syndrome. The diagnostic allows direct analysis of complex organic matrixes, like blood spots, with 100% accuracy. SLO syndrome is widespread, but the extent of its occurrence is not fully known because of the lack of a suitable screening procedure. The present method provides such a screening procedure.
2. Description of the Prior Art
Neonatal screening has become standard practice in most industrialized nations with virtually every newborn being routinely screened for phenylketonuria (PKU) and congenital hypothyroidism. Both conditions result in severe mental retardation if not diagnosed in the immediate newborn period and started on treatment. Many newborn screening programs have also added tests for additional conditions including sickle cell anemia, galactosemia, maple syrup urine disease, homocystinuria, congenital adrenal hyperplasia, biotinidase deficiency, and cystic fibrosis. The supplemental newborn screening program in place in Pittsburgh, Pa., is unique in that it offers screening for over 35 disorders using a variety of methods including acylcarnitine and amino acid profiling by tandem mass spectrometry for inborn errors of fatty acid, organic acid, and amino acid metabolism (Millington et al., "Tandem mass spectrometry: A new method for acylcarnitine profiling with potential for neonatal screening for inborn errors of metabolism ", J. Inher. Metab. Dis., Vol. 13, pp. 321-324, 1990; Chace et al., "Rapid diagnosis of phenylketonuria by quantitative analysis for phenylalanine and tyrosine in neonatal blood spots by tandem mass spectrometry", Clin. Chem., Vol. 39, pp. 66-71, 1993). Screening is generally carded out on a capillary blood specimen collected by heel stick and spotted on a filter paper card, which in turn can be mailed to a central laboratory for testing.
An interest in testing for the Smith-Lemli-Opitz (SLO) syndrome of newborns has been increasing. Clinically, the SLO syndrome is quite variable, but is generally characterized by microcephaly, growth retardation, mid-face displasia, syndactyly, polydactyly, cataracts, heart and kidney malformations, and mental retardation. Recently, a biochemical marker has been shown to be present in a large majority of SLO patients. This defect in 7-dehydrocholesterol reductase was identified (Tint et al., "Defective cholesterol biosynthesis associated with the Smith-Lemli-Opitz syndrome", N. Eng. J. Med., Vol. 330, pp. 107-113, 1994) and results in a significant reduction in blood cholesterol and a marked increase in its precursor 7-dehydrocholesterol. The frequency of the SLO syndrome has been estimated to be 1 in 20,000 newborns (Opitz et al., "Smith-Lemli-Opitz (RSH) syndrome bibliography: 1964-1993", Amer. J. Med. Genet., Vol. 50, pp. 339-343, 1994).
Techniques such as routine biochemical assays and tandem mass spectrometry have not as yet been successful (Opitz et at., "Cholesterol metabolism in the RSH/Smith-Lemli-Opitz syndrome: Summary of an NICHD conference", Amer. J. Med. Genet., Vol. 50, pp. 326-338, 1994). Currently, the preferred method of diagnosis involves capillary gas chromatography-mass spectrometry of plasma sterols following solvent extraction (Axelson, "Occurrence of isomeric dehydrocholesterols in human plasma", J. Lipid Res., Vol. 32, pp. 1441-1448, 1991). This requires derivation or chemical steps before analysis.
U.S. Pat. No. 4,224,031 by Mee et at. describes a method for analyzing organic materials in blood by chemical ionization mass spectrometry which employs a conventional mass spectrometer. The practice of the method is extraction and derivation, i.e., the use of chemical workup to carry out the analysis. In contrast, the present invention analyzes the blood spots on the filter paper directly without derivation or chemical steps.
U.S. Pat. No. 5,210,412 by Levis et al. utilizes, among other types of mass spectroscopy, a time-of-flight mass analyzer. The key point of the method is derivation of metabolites, proteins or nucleic acids with chromophores that absorb visible radiation. A two laser system is used to induce ionization. The first laser sputters the material into the vacuum and the second laser causes the ionization. In contrast, the invention of the present application utilizes a pulsed ion beam system which both sputters and ionizes the sample.
Therefore, a real and substantial need for a new analytical method that will permit detection of SLO and other additional significant and potentially treatable conditions utilizing the filter paper blood specimen remains.